Use of trehalose for treatment of neurological diseases

ABSTRACT

The present disclosure discloses trehalose for use in treatment of neurological disorders, wherein the trehalose is for a single daily administration with the daily dose between about 0.25 to about 12.5 g/kg/day. The daily dose may be about 2.67 g/kg/day. A method for treating a neurological disorders is disclosed which involves administering trehalose to a subject as a single daily administration with a daily dose between about 0.25 to about 12.5 g/kg/day and in an embodiment of this method the daily dose is about 2.67 g/kg/day. Also disclosed herein is the use of trehalose in the manufacture of a medicament for treatment of neurological disorders, wherein the trehalose is formulated as a single daily dose with the trehalose present in the medicament in an amount of between about 0.25 to about 12.5 g/kg/day. In an embodiment the daily dose is about 2.67 g/kg/day. The present disclosure provides a pharmaceutical composition for treating neurological disorders, comprising a daily dose of trehalose, and a pharmaceutically acceptable carrier wherein the daily dose of the trehalose is between about 0.25 to about 12.5 g/kg. The trehalose may be formulated as part of a foodstuff.

FIELD

The present disclosure relates to the use of trehalose for the treatmentof neurological disorders, including but not limited to Parkinson'sdisease (PD).

BACKGROUND

Autophagy is a natural, conserved, process that allows the orderlydegradation of cytoplasmic contents. There are three pathways ofautophagy, macroautophagy, microautophagy and chaperone-mediatedautophagy, of which macroautophagy is the main pathway. Autophagy playsseveral roles in cellular functioning including the breakdown andrecycling of proteins, the degradation of infectious particles and theremoval of damaged organelles, cell membranes and proteins. In certaindiseases, called proteinopathies, accumulation of structurally abnormalproteins disrupts normal cellular function. There is a wide range ofproteinopathies including many neurodegenerative disorders such asParkinson's disease, Alzheimer's disease and amyotrophic lateralsclerosis. Strategies aimed at limiting the accumulation of the abnormalproteins, such as enhancing the removal of abnormal proteins, are beinginvestigated as potential therapies for proteinopathies includingneurodegenerative disorders. For reviews on autophagy andneurodegenerative disorders see Rubinsztein et al, J Exp Med., 2015,212, pp. 979-990; Kiriyama and Nochi, Int J Mol Sci., 2015, 16, pp.26797-26812.

Trehalose is a disaccharide that represents one such strategy to reducethe accumulation of abnormal proteins. The exact mechanism of action oftrehalose is not known, however, it possesses several properties thatmay be useful in preventing neurodegeneration including stabilizingproteins, acting as a chemical chaperone for misfolded proteins andimproving the clearance of abnormal proteins (for a review see Emanuele,Curr Drug Targets, 2014, 15, pp 551-557). Trehalose reduces levels ofα-synuclein (αSYN), the protein which is misfolded in PD and drives thepathophysiology of the disease (for review see Wang and Hay, FrontNeurosci., 2015, 9, pp 1-8), in rodent models of PD (Tanji et al.,Biochem Biophys Res Commun., 2015, 465, pp746-752; He et al., MolNeurobiol., 2015, DOI 10.1007/s12035-015-9173-7; Wu et al.,Neuroscience, 2015, 284, pp 900-911).

There are several published papers showing the benefit of trehalose inanimal models of PD when the trehalose is dissolved in the animaldrinking water such that it is constantly available to the animals.These are summarized in Table 1 below.

TABLE 1 Trehalose Model administered Effect Reference MPTP- 2% trehalosein Neuroprotection Sarkar et al., lesioned drinking water observedNeurotoxicology, 2014, mouse 44, 250-262. Rotenone- 2% trehalose inNeuroprotection Wu et al., lesioned rat drinking water observedNeuroscience, 2015, 284, pp 900-911 AAV 2 and 5% in Neuroprotection Heet al., Mol α-synuclein drinking water observed Neurobiol., 2015, DOIrat 10.1007/s12035-015- 9173-7. Chronic 1% in the No behaviouralFerguson et al., Behav MPTP- drinking water effect. Some Brain Res.,2015, 292, lesioned effect on pp 68-78. mouse dopamine levels Transgenic2% trehalose in Increased Tanji et al., Biochem α-synuclein drinkingwater autophagy Biophys Res Commun., mouse 2015, 465, pp746-752.

These studies demonstrate the potential of trehalose as a treatment forPD. However, the most efficacious method of administering trehalose isunknown and uninvestigated with trehalose administered in the drinkingwater in all these studies. Similarly, whilst there are several papersin the literature showing a positive effect of trehalose in animalmodels of other neurodegenerative proteinopathies (e.g. Alzheimer'sdisease, amyotrophic lateral sclerosis and Huntington's disease)trehalose was administered in the drinking water.

Canadian Patent No. CA2608198 A1 (Lindquist et al., 2006) discloses amethod of inhibiting α-synuclein-mediated cellular toxicity bycontacting a cell expressing a toxicity-inducing amount or form ofα-synuclein with an effective amount of an osmolyte, in which theosmolyte is trehalose.

As noted above, in all the previous work performed in animal models ofPD, trehalose has been administered to animals as a solution in theirdrinking water. Furthermore, no fully quantitative methods have beenused to measure trehalose levels in either the plasma or brain in thesestudies.

Determining the best dosage regime and methodology for delivery of thetrehalose to give the most efficacious treatment of neurologicaldisorders would be very beneficial. With a rapidly aging population, andwith the cost of health care treatment increasing rapidly as a result,finding an economical and simple method of treating a wide variety ofneurological disorders, including PD, would be very advantageous.

SUMMARY

The present disclosure provides a pharmaceutical kit comprising:

-   -   trehalose for treatment of neurological disorders, and    -   instructions for a single daily administration of the trehalose        with the daily dose being between about 0.25 to about 12.5        g/kg/day.

A preferred range of trehalose is between about 0.5 to about 10g/kg/day. A more preferred range of trehalose is between about 0.75 toabout 7.5 g/kg/day. A more preferred amount is about 2.67 g/kg/day.

Disclosed herein is trehalose for use in the treatment of neurologicaldisorders, wherein the trehalose is administered as a single dailyadministration with the daily dose between about 0.25 to about 12.5g/kg/day. In an embodiment the daily dose is about 2.67 g/kg/day.

Disclosed herein is a method for treating neurological disorders,comprising:

administering trehalose to a subject as a single daily administrationwith a daily dose between about 0.25 to about 12.5 g/kg/day. In anembodiment of this method the daily dose is about 2.67 g/kg/day.

Also disclosed herein is the use of trehalose in the manufacture of amedicament for treatment of neurological disorders, wherein thetrehalose is formulated as a single daily dose with the trehalosepresent in the medicament in an amount of between about 0.25 to about12.5 g/kg/day. In an embodiment the daily dose is about 2.67 g/kg/day.

The present disclosure provides a pharmaceutical composition fortreating neurological disorders, comprising a daily dose of trehalose,and a pharmaceutically acceptable carrier wherein the daily dose of thetrehalose is between about 0.25 to about 12.5 g/kg. In an embodiment thedaily dose is about 2.67 g/kg/day.

The present disclosure provides a “foodstuff”, “food supplement”,“beverage” or “beverage supplement” composition, where “foodstuff”,“food supplement”, “beverage” or “beverage supplement” have normalmeanings for those terms and are not restricted to pharmaceuticalpreparations, for treating neurological disorders, comprising a dailydose of trehalose, and a dietary acceptable carrier wherein the dailydose of the trehalose is between about 0.25 to about 12.5 g/kg. In anembodiment of the daily dose is about 2.67 g/kg/day. Examples of“foodstuffs”, “food supplements”, “beverages” or “beverage supplements”include, but are not limited to, processed foods, ingredients added toprepared foodstuffs and beverages (e.g. cooking ingredients,sweeteners), energy bars, baked goods, protein-shakes, soft-drinks andalcoholic drinks.

The present disclosure provides a “Medical food” as defined in the Foodand Drug Administration's 1988 Orphan Drug Act Amendments for treatingneurological disorders, comprising a daily dose of trehalose, and adietary acceptable carrier wherein the daily dose of the trehalose isbetween about 0.25 to about 12.5 g/kg. In an embodiment of the dailydose is about 2.67 g/kg/day. Medical foods are foods that are speciallyformulated and intended for the dietary management of a disease that hasdistinctive nutritional needs that cannot be met by normal diet alone.

Medical foods are distinct from the broader category of foods forspecial dietary use and from traditional foods that bear a health claim.In order to be considered a medical food the product must, at a minimum(i) be a food for oral ingestion or tube feeding (nasogastric tube),(ii) be labeled for the dietary management of a specific medicaldisorder, disease or condition for which there are distinctivenutritional requirements, and (iii) be intended to be used under medicalsupervision.

The neurological disorders are any one or combination of PD,synucleinopathies and proteinopathies. The synucleinopathies are any oneof PD with dementia, dementia with Lewy bodies, MSA, essential tremor,Gaucher disease and other lysosomal storage disorders, andneurodegeneration with brain iron accumulation. The proteinopathiesinclude Alzheimer's disease, cerebral β-amyloid angiopathy, retinalganglion cell degeneration in glaucoma, prion diseases, tauopathies,frontotemporal lobar degeneration, FTLD-FUS, amyotrophic lateralsclerosis (ALS), Huntington's disease and other triplet repeatdisorders, familial British dementia, familial Danish dementia,hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexanderdisease, seipinopathies, familial amyloidotic neuropathy, serpinopathiesand retinitis pigmentosa with rhodopsin mutations.

Also disclosed herein is the use of trehalose in the manufacture of amedicament for treatment of neurological disorders, wherein thetrehalose is formulated as a single daily dose with the trehalosepresent in the medicament in an amount of between about 0.25 to about12.5 g/kg/day.

The present disclosure provides a pharmaceutical composition fortreating neurological disorders, comprising a daily dose of trehalose,and a pharmaceutically acceptable carrier wherein the daily dose of thetrehalose is between about 0.25 to about 12.5 g/kg.

A further understanding of the functional and advantageous aspects ofthe invention can be realized by reference to the following detaileddescription.

BRIEF DESCRIPTION OF DRAWINGS

The following is a description of the use of trehalose for treatment ofneurological diseases, reference being had to the accompanying drawings,in which:

FIG. 1 shows the timecourse of trehalose exposure in the plasma andbrain of rats following 1 and 7 days oral administration of trehalose(2.67 g/kg/day, p.o. administered as a single bolus dose).

FIG. 2 shows the timecourse of trehalose exposure in the plasma ofmacaques following 1 and 7 days oral administration of trehalose (2.67g/kg/day and 5.34 g/kg/day, p.o. administered as a single bolus dose).

FIG. 3 shows the comparative timecourse of trehalose exposure in theplasma of rats and macaques following 1 and 7 days oral administrationof trehalose (2.67 g/kg/day, p.o. administered as a single bolus dose).

FIG. 4 shows the correlation between trehalose levels in the CSF andbrain of macaques following 17 weeks of oral administration of trehalose(2.67 g/kg/day, p.o. administered as a single bolus dose).

DETAILED DESCRIPTION

Various embodiments and aspects of the disclosure will be described withreference to details discussed below. The following description anddrawings are illustrative of the disclosure and are not to be construedas limiting the disclosure. Numerous specific details are described toprovide a thorough understanding of various embodiments of the presentdisclosure. However, in certain instances, well-known or conventionaldetails are not described in order to provide a concise discussion ofembodiments of the present disclosure.

As used herein, the terms “comprises” and “comprising” are to beconstrued as being inclusive and open ended, and not exclusive.Specifically, when used in the specification and claims, the terms“comprises” and “comprising” and variations thereof mean the specifiedfeatures, steps or components are included. These terms are not to beinterpreted to exclude the presence of other features, steps orcomponents.

As used herein, the term “exemplary” means “serving as an example,instance, or illustration,” and should not be construed as preferred oradvantageous over other configurations disclosed herein.

As used herein, the terms “about” and “approximately” are meant to covervariations that may exist in the upper and lower limits of the ranges ofvalues, such as variations in properties, parameters, and dosage rangesto give a few examples.

As use herein, the word “trehalose” refers to the molecule shown inFormula 1 below.

Trehalose is also known by other names including “α,α-trehalose”;“α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside”. Its IUPAC name is“(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxane-3,4,5-triol”.The CAS no. of trehalose is 99-20-7 (anhydrous) 6138-23-4 (dehydrate).

The present disclosure will be illustrated using the followingnon-limiting examples/studies.

Studies

In the following examples these abbreviations are used: h=hours;min=minutes; p.o.=per oro (by mouth); t.i.d.=ter in die (3 times daily);s.e.=standard error; EV=empty vector; αSYN=alpha synuclein;TH^(+ve)=tyrosine hydroxylase positive; w/v=weight/volume;AP=anterior/posterior; DV=dorsal/ventral; AAV1/2=adeno-associated virus1/2; LC-MS/MS=liquid chromatography tandem mass spectrometry; BLQ=belowlimit of quantification; C_(max)=The peak concentration of a drug afteradministration; T_(max)=time to reach maximum concentration; AUC=areaunder the curve; t^(1/2)=elimination half-life; CSF=cerebrospinal fluid.

EXAMPLE 1

The following example shows that trehalose is more efficacious atreducing parkinsonian symptoms in a rodent model of PD when trehalose isadministered as a single, oral administration (2.67 g/kg/day) comparedto when the same amount of trehalose is administered as three separatedoses administered 8 h apart (0.89 g/kg/t.i.d.) or provided ad libitumin the drinking water (2% trehalose w/v in sterile water).

Thirty female, Sprague-Dawley rats (280-325 g) split into 5 groups(N=4-8 rats/group) and received a unilateral injection in to thesubstantia nigra (at co-ordinates AP −5.2 mm; ML −2 mm relative toBregma, DV −7.5 mm relative to skull over SN) of AAV1/2 delivering A53Talpha-synuclein (AAV1/2 αSYN) or empty vector (control). The A53T αSynexpressed is of the human sequence. Its control is an empty AAV1/2vector of the same serotype and viral construction. Both AAV1/2 vectorswere diluted in sterile phosphate buffered saline and administered at avolume of 2 μl. The concentration of AAV1/2 used was 1.7×10¹² genomicparticles/ml, which produces significant behavioural and dopaminergicnigrostriatal deficits between 3 and 6 weeks following surgical delivery(Koprich et al., PLoS One, 2011, DOI 10.1371/journal.pone.0017698).

Commencing on the day of surgery and continuing for 6 weeks, ratsreceived either vehicle (sterile drinking water) or trehalose (2.67g/kg/day) administered either in the drinking water (2% w/v), as threeseparate doses administered 8 h apart (0.89 g/kg/t.i.d., p.o.) or as asingle administration (2.67 g/kg/day, p.o).

Animal behavior was assessed pre-surgery and at 3 and 6 weekspost-surgery. Behaviour was assessed by the cylinder test to assessforelimb asymmetry with asymmetry indicating an imbalance in striataldopaminergic function between the side injected with AAV1/2 αSYN and thecontralateral side. Compounds that normalize the forelimb asymmetry arepotentially useful for treating PD. After 6 weeks of trehalosetreatment, the animals were killed approximately 30 mins after the lastadministration of trehalose. Striatal tissue from both hemispheres wascollected and analysed for dopamine levels. Substantia nigra tissue wascollected and analysed for the number of TH^(+ve) cells and the amountof A53T αSYN measured. Plasma and brain (cerebellum) samples werecollected and trehalose levels analysed by LC-MS/MS.

Rats receiving A53T αSYN exhibited an increased asymmetry, andindication of parkinsonism, as measured by the cylinder test on both Day21 and Day 42 compared to rats receiving EV (Table 2). Administration oftrehalose as a single daily oral administration (2.67 g/kg/day, p.o) for21 or 42 days reduced the asymmetry compared to mice receiving A53T αSYNalone to a level similar to rats receiving EV (Table 2). When the samedaily dose of trehalose (2.67 g/kg/day) was administered as 3 doses, 8 hapart (0.89 g/kg/t.i.d., p.o) or provided ad libitum as a 2% w/vsolution in the drinking water, there was no significant change in thelevel of asymmetry on either day 21 or day 42 (Table 2).

TABLE 2 Effect of A53T αSYN and trehalose on forelimb asymmetry in rats% asymmetry (mean ± s.e.mean) Group Day 21 Day 42 Empty vector control7.7 ± 9.4 14.9 ± 7.4  A53T αSYN  51.0 ± 14.8* 52.4 ± 20.6 A53T αSYN + 2%trehalose 48.1 ± 7.0* 51.9 ± 11.9 in drinking water A53T αSYN +trehalose 38.9 ± 16.3  67.9 ± 11.1* (0.89 g/kg, 3 times per day) A53TαSYN + trehalose 12.3 ± 16.9   3.8 ± 14.5^(#) (2.67 g/kg, once per day)Mean ± s.e.mean. * = P < 0.05 vs. EV control, ^(#) = P < 0.05 vs. A53TaSYN. One-way ANOVA followed by Fisher's LSD post-hoc test.

Rats receiving A53T αSYN also exhibited a significantly lower striataldopamine level compared to rats receiving EV (Table 3). Administrationof trehalose as a single daily oral administration (2.67 g/kg/day)partially, but significantly, restored striatal dopamine levels (Table3). Administration of the same daily dose of trehalose (2.67 g/kg/day)as either 3 doses, 8 h apart (0.89 g/kg/day, t.i.d., p.o) or provided adlibitum as a 2% w/v solution in the drinking water, did notsignificantly alter striatal dopamine levels (Table 3).

TABLE 3 Effect of A53T αSYN and trehalose on striatal dopamine levels inrats Group Dopamine (ng/mg protein) Empty vector control 123.8 ± 6.8 A53T Asyn 42.4 ± 5.8* A53T αSYN + 2% trehalose  60.2 ± 11.5* in drinkingwater A53T αSYN + trehalose 47.3 ± 7.5* (0.89 g/kg, 3 times per day)A53T αSYN + trehalose  65.4 ± 7.5*^(#) (2.67 g/kg, once per day) Mean ±s.e.mean. * = P < 0.05 vs. EV control, ^(#) = P < 0.05 vs. A53T αSYN.One-way ANOVA followed by Fisher's LSD post-hoc test.

As expected the amount of A53T αSYN per TH^(+ve) neuron increased inrats receiving A53T αSYN compared to rats receiving EV (Table 4).Administration of trehalose as a single daily oral administration (2.67g/kg/day, p.o) reduced the amount of A53T αSYN per TH^(+ve) neuroncompared to rats receiving A53T αSYN alone (Table 4). Administration ofthe same daily dose of trehalose provided ad libitum as a 2% w/vsolution in the drinking water, did not significantly alter the amountof A53T αSYN per TH^(+ve) neuron compared to rats receiving A53T αSYNalone (Table 4).

TABLE 4 Effect of A53T αSYN and trehalose on A53T αSYN expression per THneuron in rats Group aSYN/TH ratio Empty vector control 0.00000 ±0.00000 A53T αSYN  0.00027 ± 0.00010* A53T αSYN + 2% trehalose indrinking water  0.00023 ± 0.00007* A53T αSYN + trehalose (2.67 g/kg,once per day) 0.00016 ± 0.00008 Mean ± s.e.mean. * = P < 0.05 vs. EVcontrol. One-way ANOVA followed by Fisher's LSD post-hoc test.Together, these data demonstrate that administering trehalose as asingle bolus dose is more efficacious at removing αSYN, maintainingstriatal dopamine levels and reducing behavioural impairments comparedto the same dose of trehalose administered as 3 separate doses 8 h apartor administered over a 24 h period in the drinking water.

When the animals were killed 30 mins post trehalose administrationplasma and brain samples were collected (rats in the 0.89 g/kg/day grouponly received one dose of trehalose (0.89 g/kg) on this day). This timecorresponds to approximately the T_(max) of orally administeredtrehalose. Trehalose plasma levels were below the limit ofquantification (BLQ) in rats receiving trehalose in the drinking water(Table 5). Trehalose was measurable in the plasma 30 mins afterreceiving trehalose (0.89 or 2.67 g/kg) by oral gavage (Table 5). A3-fold increase in the trehalose dose (0.89 g/kg to 2.67 g/kg) lead toan ˜6-fold increase in the trehalose plasma level, i.e. that increasingthe dose produced a greater than dose-proportional increase in trehaloseexposure. Trehalose levels were below the limit of detection in thebrain of rats receiving trehalose either in the drinking water orreceiving trehalose (0.89 g/kg) by oral gavage (Table 5). Trehalose wasmeasurable in the brains of rats receiving trehalose (2.67 g/kg) by oralgavage (Table 5).

TABLE 5 Plasma and brain trehalose levels after trehalose wasadministered in drinking water of via oral gavage Trehalose Plasmatrehalose Brain trehalose Group administered level (ng/ml) level (ng/g)1 2% in drinking water BLQ BLQ 2 0.89 g/kg 3 times daily 1131 ± 178ng/ml BLQ 3 2.67 g/kg once daily 6383 ± 890 ng/ml 52.8 ± 16.9 ng/ml Mean± s.e.mean. BLQ, below level of quantification

These data demonstrate that increasing the dose of trehalose produces agreater than dose-proportional increase in systemic exposure. Thisincreased systemic exposure also allows detectable levels of trehaloseto occur in the brain. The brain is the target organ of trehalose as atreatment for PD and these results provide an explanation as to why asingle bolus administration of trehalose is more efficacious compared tothe same dose of trehalose administered as three separate doses 8 hapart or administered over a 24 h period in the drinking water (Tables2-4).

EXAMPLE 2

The following example shows the plasma and brain pharmacokinetics oftrehalose on day 1 and day 7 in Sprague Dawley rats following oraladministration of trehalose (2.67 g/kg/day) for 7 days. It shows thatthe plasma and brain samples taken for bioanalysis in Example 1 (30minutes post-dose) was close to the T_(max). It also shows that plasmalevels and brain levels of trehalose quickly drop and are no longerdetectable 8 h post-dose. These results thus support and expand upon thebioanalytical data presented in Example 1.

One hundred Sprague-Dawley rats (200-260 g), female (60) and male (40),received trehalose (2.67 g/kg/day, oral gavage in sterile water) for 1or 7 days. Groups of 5 rats (3 female, 2 male) were sacrificed atpre-dose and 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 8 h and 12 hpost-dose on days 1 and 7 and plasma and brain samples collected. Plasmaand brain samples were analysed for trehalose levels using a validatedLC-MS/MS method and plasma exposure timecourses (FIG. 1) andpharmacokinetic parameters were calculated (Table 6). Brain levels oftrehalose were approximately 1% of plasma levels at all time-points(FIG. 1).

TABLE 6 Plasma and brain pharmacokinetics of trehalose following 1 and 7days oral administration of trehalose (2.67 g/kg) Plasma Brain PKparameter Day 1 Day 7 Day 1 Day 7 T_(max) (h) 0.25 0.25 0.25 0.25C_(max) (ng/ml) 8900 8336 87.0 73.0 AUC_(0-12h) (h · ng/ml) 10851 949486.8 65.3 AUC_(0-inf) (h · ng/ml) 11136 9876 97.0 76.2 t_(1/2) (h) 0.760.89 0.81 1.06

EXAMPLE 3

The following example shows the plasma and brain pharmacokinetics oftrehalose on day 1 and day 7 in female macaques following oraladministration of trehalose (2.67 and 5.34 g/kg/day) for 7 days. Itshows that in macaques, as in rats, increasing the dose of trehaloseleads to a greater that dose-proportional increase in plasma trehaloselevel. As this occurs in two mammalian species, it is likely that itwill also occur in a third mammalian species (humans). Therefore, inhumans, administration of trehalose as a single daily oral dose islikely to produce higher systemic exposure and brain levels of trehalosethan the same daily dose administered as multiple doses over 24 h andthus provide greater therapeutic benefit.

Three female macaques (3.81-4.67 kg) received trehalose (2.67 and 5.34g/kg/day, oral gavage in sterile water) for 7 days. Plasma samples werecollected pre-dose and 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h and 24 hpost-dose on days 1 and 7 and analysed for trehalose levels using avalidated LC-MS/MS method and plasma exposure time courses (FIG. 2) andpharmacokinetic parameters were calculated (Table 7).

TABLE 7 Plasma pharmacokinetics of trehalose following 1 and 7 days oraladministration of trehalose (2.67 and 5.34 g/kg) 2.67 g/kg/day 5.34g/kg/day PK parameter Day 1 Day 7 Day 1 Day 7 T_(max) (h) 0.83 0.67 0.670.83 C_(max) (ng/ml) 10918 9578 36962 22444 AUC_(0-24h) (h · ng/ml)26657 26023 78899 56483 AUC_(0-inf) (h · ng/ml) 27445 27363 80040 57172t_(1/2) (h) 1.1 1.5 1.8 2.3

In a subsequent step, the same 3 macaques were administered trehalose(2.67 g/kg) for two days. The macaques were then killed 1 hpost-administration on day 2 and terminal brain and CSF samplescollected. The brain and CSF trehalose levels were 81.3±13.8 ng/g and562±346.5 ng/ml respectively. Similar to rats, trehalose levels inmacaque brain tissue were ˜1% of the plasma level at the correspondingtimepoint.

EXAMPLE 4

The following example shows that trehalose can reduce dysfunction of thedopaminergic system in a non-human primate model of PD when trehalose isadministered as a single, oral administration (2.67 g/kg/day). Moreover,the dose of trehalose used provided exposure levels similar to theexposure that was demonstrated to be efficacious in a rodent model of PD(see Examples 1 and 3).

Twenty-five (25), female cynomolgus monkeys (Macaca fascicularis,8.0-9.3 years of age, 3.2-4.4 kg) were split into 3 groups (n=8-9/group)and received stereotaxic injection of AAV vectors,either a vectorexpressing mutant A53T human alpha-synculein(AAV1/2-A53T-alpha-synuclein) or an empty vector (AAV1/2-EV),Genedetect, Auckland, New Zealand) into the substantia nigra. The A53TaSyn expressed is of the human sequence the AAV1/2-EV is a controlvector of the same serotype and viral construction. Precise stereotaxiccoordinates for all surgeries were calculated prior to surgery fromindividual monkey MRI scans. Injections containing theAAV1/2-A53T-alpha-synuclein or empty vector were made at a speed of 0.5μl/min and a volume of 7 μl (at a viral titre of 1.7×10¹² activeparticles per ml) into 4 sites of each hemisphere of the substantianigra. Commencing on the day of surgery and continuing for 17 weeks,macaques received either vehicle (sterile drinking water) or trehalose(2.67 g/kg/day) administered as a single daily administration (2.67g/kg/day, p.o). The macaques were then killed and striatal tissue fromboth hemispheres was collected and analysed for dopamine and dopaminetransporter (DAT) levels. Substantia nigra tissue was collected andanalysed for the number of TH^(+ve) cells and the amount of αSYNmeasured. Plasma, CSF and brain (cerebellum) samples were collected andtrehalose levels analysed by LC-MS/MS.Macaques receiving A53T exhibited a significantly lower striataldopamine and DAT levels compared to macaques receiving EV (Table 8).Administration of trehalose as a single daily oral administration (2.67g/kg/day) partially, but significantly, restored striatal dopaminelevels (Table 8). Similarly, trehalose partially restored DAT levels(Table 8).

TABLE 8 Effect of A53T αSYN and trehalose on striatal dopamine and DATlevels in macaques Striatal dopamine Striatal DAT (nCi/mg Group (ng/mgprotein) tissue) Empty vector control 160.0 ± 7.2 493.7 ± 52.5 A53T αSYN 78.9 ± 13.1*** 273.7 ± 35.7** A53T αSYN + trehalose 110.0 ± 8.3** ^(#)410.7 ± 47.9 (2.67 g/kg/day) Mean ± s.e.mean. **/*** = P < 0.01 or P <0.001 cf. EV/vehicle. ^(#) = P < 0.05 cf. αSyn/vehicle, 1-way ANOVA withHolm-Sidak multiple comparisons test.Macaques receiving A53T also exhibited fewer TH^(+ve) neurons in thesubstantia nigra and an increased expression of αSYN in the striatumcompared to macaques receiving EV (Table 9). Administration of trehaloseas a single daily oral administration (2.67 g/kg/day, p.o) only slightlyprevented the loss of TH^(+ve) neurons and increase in αSYN (Table 9).

TABLE 9 Effect of A53T αSYN and trehalose on TH^(+ve) neurons in thesubstantia nigra and aSYN expression in the striatum levels in macaquesStriatal αSYN Group No. of TH+ve neurons (ng/mg protein) Empty vectorcontrol 95678 ± 7799 36394 ± 2362 A53T αSYN  59513 ± 7720*  55455 ±5120* A53T αSYN + trehalose  69084 ± 7628*  54604 ± 4486* (2.67g/kg/day) Mean ± s.e.mean. * = P < 0.01 cf. EV/vehicle. 1-way ANOVA withHolm-Sidak multiple comparisons test.

Together, these data demonstrate that trehalose can partially preventαSYN-mediated dopaminergic dysfunction though this effect is morepronounced at the nerve terminals than at the cell bodies, asdemonstrated by a larger effect on dopamine and DAT compared to theeffect on the number of TH^(+ve) neurons.

Single plasma, brain and CSF samples were collected 1 h after the finaladministration of trehalose. One-hour post-dose corresponds toapproximately the T_(max) of orally administered trehalose in macaques.The plasma, brain and CSF trehalose levels were 2449±884 ng/ml, 129±35ng/g and 33±9 ng/ml respectively. The trehalose level in the CSFsignificantly correlated with the level in the brain demonstrating thattrehalose levels in the CSF can be used to predict trehalose levels inthe brain (FIG. 4).

Results

The results for the behavioural assessment in rats on days 21 and 42 areshown in Table 2. The αSYN (PD model) group showed an increasedasymmetry compared to the empty vector (control group). The degree ofasymmetry is indicative of an imbalance in striatal dopaminergicfunction between the side the viral vector that produces αSYN wasadministered and the contralateral side. Compounds that normalize theforelimb asymmetry are potentially useful for treating PD. Treatmentwith trehalose as a single, oral administration (2.67 g/kg/day as asingle dose group) reduced the degree of asymmetry compared to micereceiving A53T αSYN alone, indicating that trehalose given as a singledaily administration was normalising behaviour. By contrast, the samedaily dose of trehalose administered either as 2% in the drinking wateror when given as three (3) separate doses, 8 h apart did not produce abeneficial effect on behaviour.

Dopamine is the neurotransmitter that is decreased both in PD and inanimal models of PD. Increasing striatal dopamine levels is a treatmentfor PD and also reverses the parkinsonism seen in this rat model of PD.Therefore, if trehalose (single administration) increases dopaminelevels it provides a rationale for how it is improving behaviour.

As can be seen from Table 3, when trehalose is given as a once daily,single administration it significantly increases dopamine levelscompared to animals receiving αSYN alone. Interestingly, when the sametotal dose of trehalose is administered either in the drinking water ordivided into three (3) times daily administrations, the increase indopamine levels is smaller and no longer significantly different fromαSYN alone.

These results support the behavioural data; trehalose given once a dayincreases dopamine levels as well as reducing the degree of asymmetry.In addition, they provide a rationale why trehalose administered inother ways (2% in the drinking water or three (3) times daily) did notimprove behaviour (they did not increase dopamine levels).

Referring to Table 4, when the amount of αSYN per TH^(+ve) neuron wasmeasured there was a clear increase in the α-synuclein per neuron in thePD model group compared to the empty vector control group. Treatmentwith trehalose once daily reduced the amount of α-synuclein per neuronby ˜40%. Trehalose in the drinking water produced only a small reductionin the amount of α-synuclein per neuron.

This result supports the hypothesis that trehalose reduces the amount ofα-synuclein. It also further supports the idea that administering allthe trehalose in one dose is more efficacious that administrating thesame amount of trehalose over a 24 hour period.

Pharmacokinetics of Trehalose in Rats

To better understand why administrating the same amount of trehalose bydifferent routes led to differences in efficacy, studies were conductedto measure blood and brain levels of trehalose following administrationof trehalose by 2% in the drinking water, three (3) times daily (0.89g/kg each dose) and once daily (2.67 g/kg/day). Plasma samples werecollected at 30 min post dose (around the T_(max)) so that rats eitherreceived whatever trehalose they had drunk in the water in the previous24 h, 0.89 g/kg or 2.67 g/kg. The results are shown in Table 5.

There are several observations of note in the results, which are listedbelow.

1. That trehalose in the drinking water did not produce any measurabletrehalose levels in the plasma and brain. Although we do not know whenthe rats last drank relative to when the blood was collected it providesan explanation of why trehalose administered in the drinking water wasnot effective.2. That trehalose (0.89 g/kg 3×/day) produced measurable levels oftrehalose in the plasma but did not produce measurable brain trehaloselevels.3. That trehalose (2.67 g/kg 1×/day) produced measurable levels oftrehalose in the plasma and the brain.4. That the peak plasma trehalose level following 2.67 g/kg was 5.6-foldhigher than the plasma trehalose level following 0.89 g/kg. That is, a3-fold increase in dose led to a 5.6-fold increase in systemic trehaloseexposure.Full Pharmacokinetic Study in Rats

A full pharmacokinetic study was performed in rats following 1 and 7days administration of trehalose (2.67 g/kg/day given as a singleadministration) and the results are summarized in FIG. 1 and Table 6.

As can be seen, trehalose is rapidly absorbed into, and rapidlyeliminated from, the plasma following oral administration. No trehaloseis found in the plasma or brain 8 h post-dose. This is important as inthe efficacy study the group receiving 3 administrations of trehalosewere given 8 h apart and so plasma trehalose levels had declined tobelow the limit of detection between each subsequent trehaloseadministration. Brain trehalose levels represent approximately 1% ofplasma levels and brain levels follow a very similar time-course toplasma levels.

Pharmacokinetics of Trehalose in Non-Human Primates

A full pharmacokinetic study was performed in macaques following 1 and 7days administration of trehalose (2.67 or 5.34 g/kg/day given as asingle administration) and the results are summarized in FIG. 2 andTable 7.

Similar to rats, trehalose is rapidly absorbed into, and rapidlyeliminated from, the plasma following oral administration to macaques.No trehalose was measurable in the plasma ˜8 h post-dose. Also similaris that a doubling of the dose of trehalose led to an approximately3-fold increase in plasma trehalose levels (as measured by C_(max) orAUC)—again showing that increasing the dose produces a greater thanexpected increase in plasma levels.

Brain and CSF levels of trehalose were also measured following 2 daysadministration of trehalose (2.67 g/kg). Brain and CSF samples werecollected 1 h post-dose (approximately the plasma T_(max)) and brain andCSF trehalose levels were 81.3±13.8 ng/g and 562±346.5 ng/mlrespectively. Therefore, the brain trehalose levels representapproximately 1% of plasma levels, which is similar to rats.

Efficacy of Trehalose in Non-Human Primates

As can be seen from Table 8 when trehalose is administered once daily tonon-human primates it significantly increases dopamine levels comparedto animals receiving αSYN alone. This result is very similar to theresult obtained in rats (compare Tables 3 and 8).

This result, along with the finding that the same dose of trehalose on ag/kg basis provides a similar plasma trehalose exposure, supports thehypothesis that a dose of trehalose that provides a similar trehaloseexposure in rats and non-human primates also significantly reducesαSYN-induced striatal dopamine loss in both species. Together, theseresults suggest that an equivalent dose, on a g/kg basis, of trehaloseproduces similar trehalose exposures in the plasma and brain of rats andnon-human primates and that this exposure also produces similarefficacious effects in rats and non-human primates. As these effects arepreserved between rats and non-human primates it is also likely thatthey will translate to humans with Parkinson's disease and this allowsus to estimate a dose of trehalose that is likely to be efficacious intreating Parkinson's disease.

Extrapolating From Animal Data to a Human Dose

On a dose per body weight basis, trehalose produced very similar plasmaand brain levels in rats and macaques (see FIG. 3 and Tables 6 and 7)when dosed with 2.67 g/kg/day. We also know that a trehalose dose of2.67 g/kg/day was efficacious in the rat and non-human primate.Therefore, it is contemplated that an efficacious dose for humansincludes 2.67 g/kg which is estimated to produce a trehalose plasmaC_(max) of ˜10000 ng/ml. From the rat data it is also clear that a doseof 0.89 g/kg will be ineffective.

Therefore, if we assume the average human weight is 80 kg then based onthe results disclosed herein, it is estimated that 214 g/day would be anefficacious dose. Similarly, it is contemplated that a dose below 71g/day will not be efficacious in human. Table 10 below shows therequired daily dosages for the various weight ranges.

TABLE 10 Body weight (kg) Required daily trehalose (g) Up to 50 130 51-60 160  61-70 190  71-80 210  81-90 240  91-100 270 101-110 300 Over110 330

The trehalose may be formulated as a pharmaceutical composition fortreating neurological disorders, comprising a daily dose of trehalose,and a pharmaceutically acceptable composition wherein the daily dose ofthe trehalose is between about 0.25 to about 12.5 g/kg. Examples ofpharmaceutical acceptable compositions include carriers, diluents,adjuvants, excipients or vehicles, preserving agents, fillers,disintegrating agents, buffering agents, penetration enhancers, wettingagents, emulsifying agents, suspending agents, sweetening agents,flavouring agents, perfuming agents, antibacterial agents, antifungalagents, lubricating agents and dispensing agents. Suitable dosage formsinclude, for example, tablets, dragees, powders, elixirs, syrups, liquidpreparations including suspensions and emulsions, lozenges, granules andcapsules. Techniques and formulations generally may be found inRemington, Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,latest edition.

The trehalose may be incorporated into a “foodstuff”, “food supplement”,“beverage” or “beverage supplement” composition, where “foodstuff”,“food supplement”, “beverage” or “beverage supplement” have normalmeanings for those terms and are not restricted to pharmaceuticalpreparations, for treating neurological disorders, comprising a dailydose of trehalose, and a dietary acceptable carrier wherein the dailydose of the trehalose is between about 0.25 to about 12.5 g/kg. In anembodiment of the daily dose is about 2.67 g/kg/day.

The present disclosure provides a pharmaceutical kit comprising:

-   -   trehalose for treatment of neurological disorders, and    -   instructions for a single daily administration of the trehalose        with the daily dose being between about 0.25 to about 12.5        g/kg/day.

In some embodiments of the pharmaceutical kit the daily dose is betweenabout 0.5 to about 10 g/kg/day.

In some embodiments of the pharmaceutical kit the daily dose is betweenabout 0.75 to about 7.5 g/kg/day.

In some embodiments of the pharmaceutical kit the daily dose is one ofbetween about 1 to about 5 g/kg/day and about 1.25 to about 3.75g/kg/day.

In an embodiment of the pharmaceutical kit the daily dose is about 2.67g/kg/day.

The pharmaceutical kit may include the following instructions:

Body weight (kg) Required daily trehalose (g) Up to 50 130  51-60 160 61-70 190  71-80 210  81-90 240  91-100 270 101-110 300 Over 110 330Translatability to Other Diseases

While the present studies have been focused on PD, it will beappreciated that for PD the studies have shown that trehaloseadministered as a single bolus dose is more efficacious than whenadministered throughout the day. It is therefore contemplated that theseresults may be applicable to diseases other than PD, especially thosediseases where it has already been demonstrated that trehaloseadministered in the drinking water is efficacious (e.g. Alzheimer'sdisease, tauopathies, amyotrophic lateral sclerosis and Huntington'sdisease). It is contemplated that there are two (2) other ‘groups’ ofdiseases, in addition to PD, that may exhibit the same results as foundwith PD, namely synucleinopathies (diseases that have misfoldedα-synuclein), and proteinopathies (diseases that have a misfoldedprotein other than α-synuclein). PD is a synucleinopathy andsynucleinopathies are a subset of proteinopathies. In summary thesediseases may include:

1. Parkinson's disease,

2. Synucleinopathies including (but not limited to), Parkinson's diseasewith dementia, dementia with Lewy bodies, MSA, essential tremor, Gaucherdisease and other lysosomal storage disorders, neurodegeneration withbrain iron accumulation, and

3. Other proteinopathies including (but not limited to), Alzheimer'sdisease, Cerebral β-amyloid angiopathy, Retinal ganglion celldegeneration in glaucoma, Prion diseases, Tauopathies, Frontotemporallobar degeneration, FTLD-FUS, Amyotrophic lateral sclerosis (ALS),Huntington's disease and other triplet repeat disorders, FamilialBritish dementia, Familial Danish dementia, Hereditary cerebralhemorrhage with amyloidosis, CADASIL, Alexander disease, Seipinopathies,Familial amyloidotic neuropathy, Senile systemic amyloidosis,Serpinopathies and Retinitis pigmentosa with rhodopsin mutations.

CONCLUSION

The present inventors have conducted studies to investigate thepotential of trehalose to be developed as a treatment for variousneurological disorders, including but not limited to Parkinson'sdisease. As part of these studies the inventors:

-   -   1) developed a quantitative method for measuring trehalose        levels in plasma and brain tissues;    -   2) investigated the efficacy of three different ways of        administrating the same dose of trehalose (in drinking water,        administered as a single dose or administered as 3 doses eight        hours apart).    -   3) investigated the plasma and brain pharmacokinetics of        trehalose following oral administration of trehalose.

In summary the inventors have demonstrated the following.

-   -   1) When trehalose was given as a single administration it was        much more efficacious than when the same amount of trehalose was        administered in the drinking water or as 3 administrations, 8        hours apart.    -   2) That blood levels of trehalose are not dose linear.        Increasing the dose 3-fold led to a 6 fold increase in trehalose        plasma level in rats and a doubling of the dose led to a greater        than 3-fold plasma exposure in macaques.    -   3) That trehalose only reached the brain in detectable amounts        when the dose of trehalose was administered as a single bolus        dose.

Taken together, these results demonstrate the very surprising resultthat trehalose is more efficacious when given as a single bolus dose.Without being bound by any hypothesis, the inventors contemplate that areason for this enhanced efficacy may be that dosing all the trehalosein one administration leads to a greater systemic drug exposure thanwould be expected if the drug exhibited linear kinetics. The reason whythis occurs is unknown but the inventors contemplate that it might bedue to higher doses of trehalose saturating the systems that eliminatetrehalose from the body.

The foregoing description of the preferred embodiments of the inventionhas been presented to illustrate the principles of the invention and notto limit the invention to the particular embodiment illustrated. It isintended that the scope of the invention be defined by all of theembodiments encompassed within the following claims and theirequivalents.

Therefore what is claimed is:
 1. A composition for treating neurologicaldisorders in human patients, comprising a single oral daily dose oftrehalose in a physiologically acceptable carrier wherein the singleoral daily dose of the trehalose is between about 100 to about 1000grams based on an 80 kilogram body weight of a human.
 2. The compositionaccording to claim 1, wherein the carrier is a pharmaceuticallyacceptable carrier.
 3. The composition according to claim 1, wherein thecarrier is foodstuff, foodstuff supplement, beverage or beveragesupplement.
 4. The composition according to claim 3, wherein the carrieris a calorific material comprising fats, oils, carbohydrates, proteins,or sources of minerals, vitamins or fiber or any combination thereof. 5.The composition according to claim 3, wherein the carrier is any one ofdairy, cereal, vegetable, meat, fish, poultry, fruit based foodstuffs,processed foods, cooking ingredients and sweeteners.
 6. The compositionaccording to claim 3, wherein the carrier is any one of water,carbonated beverages, uncarbonated beverages, fruit juices, infusiondrinks, coffee, teas, protein-shakes, soft-drinks or alcoholic drinks.7. The composition according to claim 1 wherein the single oral dailydose is between about 100 to about 800 grams.
 8. The compositionaccording to claim 1 wherein the single oral daily dose is between about100 to about 600 grams.
 9. The composition according to claim 1 whereinthe single oral daily dose is between about 100 to about 400 grams. 10.The composition according to claim 1 wherein the single oral daily doseis between about 100 to about 300 grams.
 11. The composition accordingto claim 1 wherein the single oral daily dose is about 215 grams. 12.The composition according to claim 1 wherein the neurological disordersare any one or combination of Parkinson's disease, synucleinopathies andproteinopathies.
 13. The composition according to claim 1 wherein theneurological disorder is Parkinson's disease.
 14. The compositionaccording to claim 12 wherein the synucleinopathy is any one ofParkinson's disease with dementia, dementia with Lewy bodies, MSA,essential tremor, Gaucher disease and other lysosomal storage disorders,and neurodegeneration with brain iron accumulation.
 15. The compositionaccording to claim 12 wherein the proteinopathies include Alzheimer'sdisease, cerebral β-amyloid angiopathy, retinal ganglion celldegeneration in glaucoma, prion diseases, tauopathies, frontotemporallobar degeneration, FTLDFUS, amyotrophic lateral sclerosis (ALS),Huntington's disease and other triplet repeat disorders, familialBritish dementia, familial Danish dementia, hereditary cerebralhemorrhage with amyloidosis, CADASIL, Alexander disease, seipinopathies,familial amyloidotic neuropathy, serpinopathies and retinitis pigmentosawith rhodopsin mutations.
 16. The composition according to claim 1wherein the single oral daily dose of trehalose is in the followingamount: Body weight (kg) Amount of trehalose (g) Up to 50 130  51-60 160 61-70 190  71-80 210  81-90 240  91-100 270 101-110 300 Over 110 
 330.


17. The composition according to claim 9 wherein the neurologicaldisorders are any one or combination of Parkinson's disease,synucleinopathies and proteinopathies.
 18. The composition according toclaim 9 wherein the neurological disorder is Parkinson's disease.
 19. Apharmaceutical kit comprising: the composition according to claim 1, andinstructions for a single daily administration of the composition fortreatment of neurological disorders.
 20. The pharmaceutical kitaccording to claim 8, wherein the neurological disorders are any one orcombination of Parkinson's disease, synucleinopathies andproteinopathies.